The long term objective of this project is to understand the molecular mechanisms by which the maize leaf undergoes cellular differentiation and morphogenesis. Our studies of C4 pathway genes and of their cell-specific expression during maize leaf development have indicated that cell interactions, cell-specific use of small gene family members, and both positional and lineage information play roles in the differentiation two major leaf cell types, bundle sheath (BS) and mesophyll (M). This proposal seeks to determine the molecular basis of these phenomena, with the following specific aims: (1) to find the cis- and trans-acting regulatory elements that control cell-specific and position-dependent expression of genes for NADP-dependent malic enzyme (ME), NADP-dependent malate dehydrogenase (MDH), phosphoenolpyruvate carboxylase (PEPCase), and their housekeeping counterparts. (2) to obtain and compare DNA sequences for both photosynthetic (leaf) and housekeeping (all tissue) members of ME and MDH gene families and to compare sequences of ME, MDH, and other C4 pathway genes regulated in the same way, and (3) to investigate the dependence of BS and M cell differentiation on cell position, cell lineage, and local cell interactions in the leaf, using C4 genes as cell markers in in situ RNA and protein studies, analysis of leaf pattern mutants, and analysis of irradiated or cultured leaf primordia. Our recent development of methods for culture of maize leaf primordia and the regeneration of plants from isolated shoot apices will permit novel approaches to the study of patterned gene expression in maize, including cell-tagging. We will rely on previously prepared C4 antibody, cDNA, and gene probes for the studies on C4 gene function and leaf cellular differentiation.